(1) Field of the Invention
The present invention relates to a method for performing an immunoassay for detecting an analyte which uses colored polystyrene latex particles for detecting the analyte. The method is particularly useful for detecting microorganisms which produce extracellular polysaccharides (EPS) also known as exocellular polysaccharides, capsule, and/or lipopolysaccharides (LPS). In a preferred method for detecting microorganisms which produce EPS, LPS, or both, the EPS and/or LPS is extracted from a sample with cetyltrimethylammonium bromide (CTAB) to produce molecular aggregates which are then preferentially bound to colored polystyrene latex particles over other components in the sample, and the bound EPS and/or LPS detected using a lateral flow immunoassay apparatus which has immobilized thereon antibodies specific for the EPS and/or LPS. The method can also be used to detect particular viruses, for example viruses of the potyviridae or tobamoviridae group.
(2) Description of Related Art
Bacterial plant pathogens cause many important diseases of field and orchard crops and produce damages worth many millions of dollars (Goto, In: Fundamentals of Bacterial Plant Pathology, Academic Press, New York, N.Y. (1990)). Some of these pathogens have been present in U.S. for many years. Others have not been detected in this country; however, their introduction may produce quarantine actions and cause serious economic loss. During the month of August, 2002 USDA-APHIS released a list of potential pathogens which could be used for agricultural bioterrorism. Among the nine pathogens listed, four are bacterial plant pathogens such as Ralstonia solanacearum (Rs) race 3.
Traditional diagnosis of bacterial plant pathogens involved isolating bacteria from an infected sample using selective media and studying its morphological, nutritional, and biochemical characteristics (Schaad et al., In: Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3rd Edition. APS Press, St. Paul, Minn. (2000)). Several analytical procedures based on nutritional (MICROLOG Microbial Identification System, Biolog Inc., Hayward, Calif.) and fatty acid analysis are commercially available. However, these procedures require trained personnel and in many cases, the data turn-around time is considerable.
New molecular biology techniques such as polymerase chain reaction (PCR) and real-time PCR offer specific and sensitive diagnosis. However these procedures are expensive, require specialized equipment, lengthy sample processing protocols, and skilled personnel. Real-time PCR with molecular beacons (Tyagi and Kramer, Nature Biotecnol. 14: 303-308 (1996)) and nucleic acid sequence-based amplification (NASBA) (Compton, Nature. 350: 91-92 (1991)) are some of the newest DNA-based assays which are rapid and sensitive. While such molecular diagnostics are effective, in practice they require a high level of expertise, demanding sample extraction methodologies, and a generous diagnostic budget. An enrichment PCR assay, called “BIO-PCR”, shows greater sensitivity than direct PCR (Schaad et al., Plant Dis. 83: 1095-1100 (1999)) when used to detect pathogen from plant samples. However, the principle of sample enrichment by culturing in a media also enhances the sensitivity of serological tests. For example, Hoszowski et al., Int. J. Food Microbiol. 28: 341-350 (1996) were able to detect as few as five colony-forming units (pre-enrichment number) of Salmonella from 100 mL of chicken carcass rinsing with a filtration, enrichment, and colony blot immunoassay technique. A rational approach may be testing a higher volume of samples using a rapid inexpensive diagnostic test and confirming positives with PCR by sending the sample to a diagnostic lab.
Serological tests such as traditional agar double-diffusion assays and more recently, enzyme linked immunosorbent assay (ELISA), immunoblots, immunofluorescence (IF), and lateral flow immunoassays (LFA) have also been used extensively for diagnosis of plant bacterial pathogens (Alvarez, In: Plant Pathogenic Bacteria, 3rd Edition. Schaad et al., Eds. APS Press, St. Paul, Minn. (2000), pp. 338-342). In general, these tests have been of limited use because of their lack of specificity and sensitivity and cross-reactivity to other bacterial species. Older serological tests utilizing polyclonal antibodies made against bacterial proteins are of limited utility due to their cross reactivity (Robinson-Smith et al., Food and Agricultural Immunol. 7: 67-79(1995)) and specificity (Hampton, In: Serological Methods for Detection and Identification of Viral and Bacterial Plant Pathogens, a Laboratory Manual. APS Press, St. Paul, Minn. (1990)). The invention of monoclonal antibody technology by Kohler and Milstein in 1975 stimulated rapid progress in serological techniques. Hybridoma technology has been used to generate and characterize monoclonal antibodies (MAbs) specific for several species of bacterial plant pathogens (Alvarez et al., Phytopathol. 75: 722-728 (1985); Alvarez et al., Phytopathol. 81: 857-865 (1991); Alvarez et al., In: Bacterial Wilt International Conf., Kaohsiung, Taiwan, ACIAR Proc. No. 45. (1992), pp. 62-69; Alvarez et al., Plant Pathology 45: 358-366 (1996); Alvarez et al., In: Seed Health Testing: Progress Towards the 21st Century. Hutchins and Reeves, Eds. CAB International, Wallingford, United Kingdom (1998), pp. 175-183; Alvarez et al., In: Proc. 3rd. International Seed Testing Association, Seed Health Symposium, Iowa State University, Ames (1999), pp. 110-114; Jordan, In: Molecular Methods in Plant Pathology, Singh, Ed., Lewis Publishers, Inc., Boca Raton, Fla. (1995), pp. 395-412 (1995); Hampton et al., Serological Methods for Detection and Identification of Viral and Bacterial Plant Pathogens, a Laboratory Manual. APS Press, St. Paul, Minn. (1990); Torrance, Euro. J. Plant Pathol. 101: 351-363 (1995); Wong, LETT. APPL. M. 10: 241-244 (1990). Thus, by using hybridoma technology, monoclonal antibodies have been produced which can differentiate bacterial strains, races, and biovars within the same genus (Alvarez and Bennedict, In: Methods in Phytobacteriology. Klement et al. Eds. Akademiai Kiado Budapest (1990), pp. 180-185; Goto, In: Fundamentals of Bacterial Plant Pathology, Academic Press, New York (1990)). Thus, immunodiagnostic techniques which use monoclonal antibodies (MAbs) are now used for detection of pathogenic bacteria both from seed (Alvarez and Kaneshiro, In: Proc. 3rd Intl. Seed Testing Assoc., Seed Health Symp., Iowa State University, Ames, Iowa (1999), pp. 93-97; Alvarez et al., In: Seed Health Testing: Progress Towards the 21st Century, Hutchins and Reeves, Eds., CAB International, Wallingford, UK (1997), pp. 175-183)) and other plant materials (Baer and Gudmestad, Phytopathol. 83: 157-163 (1993); Gitaitis et al., Plant Dis. 75: 834-838 (1991); McLaughlin and Chen, In: Serological Methods for Detection and Identification of Viral and Bacterial Plant Pathogens, a Laboratory Manual. Hampton et al., Eds., APS Press, St. Paul, Minn. (1990), pp. 197-205.
Lateral flow immunostrip assays have several advantages over other currently available formats such as simple to use, portable, inexpensive, stable, and have longer shelf-life. There is extensive art in the field of lateral flow immunostrip technology which is exemplified by the following patents: U.S. Pat. No. 6,391,652 B1 to Okada et al.; U.S. Pat. No. 6,368,875 to Geisberg; U.S. Pat. No. 6,352,862 B1 to Davis et al.; U.S. Pat. No. 6,342,396 B1 to Perrin et al.; U.S. Pat. No. 6,228,660 B1 to May et al.; U.S. Pat. No. 6,180,417 B1 to Hajizadeh et al.; U.S. Pat. No. 5,989,921 to Charlton et al.; U.S. Pat. No. 5,965,458 to Kouvonen et al.; U.S. Pat. No. 5,877,028 to Chandler et al.; U.S. Pat. No. 5,827,749 to Akers, Jr.; U.S. Pat. No. 5,814,407 to Richard et al.; U.S. Pat. No. 5,766,961 to Pawlak et al.; U.S. Pat. No. 5,770,460 to Pawlak et al.; U.S. Pat. No. 5,741,662 to Madsen et al.; U.S. Pat. No. 5,716,778 to Weng et al.; U.S. Pat. No. 5,712,172 to Huang et al.; U.S. Pat. No. 5,712,170 to Kouvonen et al.; U.S. Pat. No. 5,695,928 to Stewart; U.S. Pat. No. 5,686,315 Pronovost et al.; U.S. Pat. No. 5,654,162 to Guire et al.; U.S. Pat. No. 5,620,845 to Gould et al.; U.S. Pat. No. 5,591,645 to Rosenstein; U.S. Pat. No. 5,498,551 to de Jaeger et al.; U.S. Pat. No. 5,489,537 to Van Aken; U.S. Pat. No. 5,437,983 to Watts et al.; U.S. Pat. No. 5,424,193 to Pronovost et al.; U.S. Pat. No. 5,415,994 to Imrich et al.; U.S. Pat. No. 5,266,497 to Imai et al.; U.S. Pat. No. 5,252,459 to Tarcha et al.; U.S. Pat. No. Re. 34,405 to Gould et al.; U.S. Pat. No. 5,238,652 to Sun et al.; U.S. Pat. No. 5,225,322 to Wolf; U.S. Pat. No. 5,212,061 to Snyder et al.; U.S. Pat. No. 5,096,837 Fan et al.; U.S. Pat. No. 5,075,078 to Osikowicz et al.; U.S. Pat. No. 5,030,561 to Mapes et al.; U.S. Pat. No. 5,028,535 to Buechler et al.; U.S. Pat. No. 4,954,452 to Yost et al.; U.S. Pat. No. 4,952,520 to Okusa et al.; U.S. Pat. No. 4,943,522 to Eisinger et al.; U.S. Pat. No. 4,920,046 to McFarland et al.; U.S. Pat. No. 4,861,711 to Friesen et al.; U.S. Pat. No. 4,855,240 to Rosenstein et al.; U.S. Pat. No. 4,837,168 to de Jaeger et al.; U.S. Pat. No. 4,703,017 to Campbell et al.; U.S. Pat. No. 4,663,277 to Wang; U.S. Pat. No. 4,639,425 to Baier; U.S. Pat. No. 4,435,504 to Zuk et al.; U.S. Pat. No. 4,415,700 to Batz et al.; U.S. Pat. No. 4,376,110 to David et al.; U.S. Pat. No. 4,313,734 to Leuvering; U.S. Pat. No. 4,187,075 to Nöller; U.S. Pat. No. 4,168,146 to Grubb et al.; and European Patent Application No. EP0810436 to Davis et al.
It has been recognized that a preferred assay for detecting microorganisms and viruses, in particular, microorganisms which produce extracellular polysaccharides (EPS) and/or lipopolysaccharides (LPS), would be portable and simple to use such that no special training or equipment would be required to perform the assay. However, in spite of recent improvements in diagnostic methods for detecting microorganisms and viruses, a rapid, field-based diagnostic method for detection of EPS and/or LPS produced by microorganisms as an indicator of infection by the microorganism is still lacking, in particular, an assay for detecting a microorganism which does not kill the microorganism. Such an assay would be particularly useful for detecting microorganisms which infect plants and microorganisms which cause systemic infections in animals or humans.